We have been studying crystallins, the abundant water-soluble proteins responsible for the optical properties of the eye lens, as a model of tissue-specific gene expression. In FY2004 we have accomplished the following: We have continued to examine the mouse small heat shock protein (shsp)/ alphaB-crystallin gene locus. A shsp/alphaB-crystallin-related gene (MKBP/HSPB2), which was derived by duplication of the shsp/alphaB-crystallin gene during evolution, is present 1 kb upstream of the mouse shsp/alphaB-crystallin gene and is transcribed in the opposite direction. The MKBP/HSPB2 gene is expressed in muscle and heart, but not in lens. In FT2004 we have confirmed the presence of a negative regulatory element (-836/-629) between the enhancer and the MKBP/HSPB2 gene. Mutagenesis of 2 conserved E-boxes (often associated with suppression) present in the silencing regions decreased (rather than increased) alphaB-crystallin promoter activity without affecting the nearby Mkbp/hspB2 promoter. Factors which bind the alphaB-crystallin enhancer elements (E1-E5) are being isolated. Earlier studies implicated HSF, SRF and MyoD. In FY2004 we showed that the glucocorticoid receptor (GR) and Sp1 bind enhancer elements E1 and E3, respectively, and differentially affect the divergently arranged promoters. We have continued our collaborative project with Dr. Eviatar Nevo (University of Haifa, Israel) on the blind mole rat. This rodent develops an eye during embryogenesis that regresses and has a degenerate lens fragment at best in the adult. We showed earlier that the alphaB-crystallin promoter/enhancer of the mouse and blind mole rat are similar but not identical. Luciferase reporter transgenes driven by either a mole rat or a mouse alphaB-crystallin promoter/enhancer are expressed similarly in the heart of transgenic mice. However, the mole rat promoter/enhancer is at least than 10 times more active than the mouse promoter/enhancer in skeletal muscle and barely active in lens in the transgenic mice. The mole rat promoter does function during early embryonic in the transgenic mouse, but turns off later in development, suggesting the existence of a developmental switch for shsp/alphaB-crystallin gene expression. We concluded that the shsp/alphaB-crystallin promoter/enhancer underwent adaptive changes corresponding to the subterranean evolution of the blind mole rat. In FY2004 we provided evidence that a mole rat promoter motif, possibly associated with a Pax3-binding sequence, may contribute to the high muscle activity of the promoter in transgenic mice, but absence of this candidate Pax3-binding site in the mouse promoter is not sufficient to explain high lens activity. In FY2004 we have been examining shsp/alphaB-crystallin gene expression in zebrafish. Two alphaB-crystallin genes were found and both are expressed in the lens, however with considerably different temporal regulation. Both of the zebrafish alphaB-crystallin genes have been cloned. The molecular basis for their expressions in lens and other tissues of the zebrafish is under investigation. Finally, the roles of aldehyde dehydrogenase 1a1 (ALDH1a1) and ALDH3a1 were studied in collaboration with Dr. Vasilis Vasiliou (University of Colorado Health Sciences Center, Denver) and Dr. Naseem Ansari (University of Texas Medical Center, Galveston, TX). Double ALDH3a1 and ALDH1a1 knockout mice were created. Their lenses are susceptible to UV-induced opacities, although their corneas appear normal, consistent with our recent collaborative findings that suppression of ALDH1a1 expression in rat lenses leads to opacities. The double knockout mice provide useful tools to study oxidative defenses of the lens and cornea.